Analysis of Monobutyryl and Dibutyryl Derivatives of Adenosine 3′,5′-Monophosphate in Biological Samples Using Isocratic Ion Pair High-Performance Liquid Chromatography

Abstract

Adenosine 3′,5′-monophosphate (cyclic AMP), its dibutyryl and monobutyryl derivatives, and a number of other naturally occurring adenine-containing compounds were separated by isocratic ion pair high-performance liquid chromatography. A mobile phase consisting of 30% methanol in 0.1 M KH2PO4 (pH 4.0) containing 1mM tetramethylammonium hydroxide a6s the counterion was used to separate the butyryl derivatives. To sufficiently separate cyclic AMP from other adenine-containing compounds, a mobile phase containing 6% methanol in the same aqueous buffer plus counterion was used. Extraction of these cyclic nucleotides from deproteinized biological samples using disposable reverse-phase extraction columns is described. This not only eliminated lipophilic contaminants, but also served to concentrate the samples. The outlined procedures were used to determine the concentrationns of the butyryl derivatives in lung tissue and perfusate following a 35-min lung perfusion with 100 μΜ N6-O2′-dibutyryl cyclic AMP. The role of this technique in the analysis of cyclic nucleotide derivatives as compared with conventional assay procedures is discussed.